Pre-steady state kinetics and O2/NO- reactivity among cysteine dioxygenase enzymes Abstract. Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O2- dependent oxidation of L-cysteine (Cys) to produce cysteine sulfinic acid (CSA). This enzyme catalyzes the first committed step in Cys catabolism, thus it is central to mammalian sulfur metabolism and redox homeostasis. Enzymes involved in sulfur-oxidation and transfer are increasingly being recognized as potential drug targets for development of antimicrobials, therapies for cancer, and inflammatory disease. Imbalances in Cys metabolism have also been identified in a variety of other neurological disorders (motor neuron disease, Parkinson's, and Alzheimer's). These observations suggest a potential correlation between impaired sulfur metabolism, oxidative stress, and neurodegenerative disease. Ironically, while sulfur is considered one of the six primordial elements required during the early stages of biological evolution, mechanistic characterization of enzymes involved in sulfur metabolism is far from complete. In principle, a greater understanding of how sulfur-oxidizing enzymes deviate mechanistically between mammals and pathogenic organisms could lead to the design of specific antimicrobial and antineoplastic agents without inadvertently disrupting native sulfur metabolism. In contrast to the canonical 2-His-1-carboxylate facial triad motif exhibited by nearly all non-heme mononuclear iron enzymes, the active site of mammalian CDO is comprised of a neutral 3-His facial triad. Additionally, a covalently cross-linked cysteine-tyrosine pair (C93-Y157) has been identified directly adjacent to the ferrous iron active site. Given the substantial deviations observed in the first- and outer- Fe coordination sphere of this enzyme, we hypothesize that the reactivity and thus transient intermediates produced during thiol-oxidation in native catalysis will significantly deviate from those observed in 2-His-1-carboxylate containing non-heme iron enzymes. This proposal represents a continuation of our ongoing efforts to develop a chemical mechanism for the thiol dioxygenase subset of non-heme iron enzymes. We provide substantial evidence indicating that the substrate-bound Mus musculus CDO (Mm CDO) exhibits unique reactivity with nitric oxide (NO) resulting in an usual electronic structure for the {FeNO}7 (S = 1/2) ternary complex. Moreover, LC-MS/MS product analysis of NO-reactions with substrate-bound Mm CDO identifies fragmentation ions consistent with formation of a sulfur-nitrogen bond, suggesting that the product generated in these reactions is cysteine sulfinamide. To our knowledge this `dioxygenase'-like reactivity with NO has not previously been observed for any other non-heme mononuclear iron enzyme. Interestingly, this reaction only occurs in the mammalian enzyme. Equivalent reactions prepared from the substrate-bound Azotobacter vinelandii CDO (Av CDO) show reversible NO-binding to produce the more typical {FeNO}7 (S = 3/2) ternary complex without formation of the putative cysteine sulfinamide product. Specific Aim 1 outlines a general strategy to characterize this unusual chemistry. These studies will also prove insight into the physiologically relevant reactions with molecular oxygen among this class of enzymes. Interrogation of the relative timing of chemical and non-chemical steps during CDO catalysis will be invaluable for validating the chemical mechanism for this enzyme. Specific Aim 2 outlines the development of a kinetic mechanism for CDO by pre-steady state kinetic methods. Preliminary single-turnover kinetics and stopped-flow results are provided to demonstrate the feasibility of direct interrogation of transient enzyme species during catalytic turnover. These experiments will be used to expand our preliminary kinetic mechanism obtained from steady-state analysis. These efforts are synergistic with our overreaching objective to trap and spectroscopically characterize [Fe-O] intermediates by rapid-mix freeze-quench EPR/Mssbauer techniques. NIH support of this AREA proposal will significantly contribute to UTA's strategic plan of becoming a national research university and a source of well-prepared STEM human resources. Moreover, the interdisciplinary scope of research proposed will provide unique training opportunities for students interested in research at the interface of chemistry and biology. Students at all levels receive `hands-on' training in the preparation and handling of complex biomolecules (DNA and proteins), molecular biology (cloning, expression, site-directed mutagenesis), bioinorganic enzymology (steady-state and pre-steady- state kinetics), handling of redox-sensitive materials, as well as analytical/spectroscopic methods (HPLC, LC-MS, UV-visible, EPR).